Mass spectrometry is one of the most important tools for proteomics. Especially important are new protocols recently implemented for the identification of low- concentration analytes in the presence of very large backgrounds. NEMS-based mass spectrometry (NEMS-MS) can provide complementary and powerful new assays for extremely rare or dilute analytes. If successful, the proposed program will culminate in prototype demonstrations of new methods for biological mass spectrometry providing unprecedented resolution - carried out in close collaboration with the world's preeminent leaders in the field of proteomic mass spectrometry. The application below charts a methodical course toward the realization of single-molecule NEMS-MS. We have shown theoretically that the intrinsic resolution of nanoelectromechanical systems (NEMS) -based mass detectors is well below 1 Da. For the field of biological mass spectrometry (MS) the implications of this are profound. To follow-on from our successful R21-funded pilot program - in which we have achieved the first demonstration of single-molecule NEMS mass detection in real time - we propose a 5- year research and development program (R01) to develop compact, next-generation, high-throughput mass spectrometers with single-molecule resolution. These will be based on the large-scale integration of microfluidic-interfaced NEMS. PUBLIC HEALTH RELEVANCE: Proteomics is the study of the biological machinery that underlies all life processes - and it is key to biomedical and pharmaceutical research. The paramount tool for protein identification is mass spectrometry (MS), but existing tools typically work only if hundreds of millions of identical molecules can be extracted from cell cultures for analysis. This project proposes to develop a new technique from nanoscience, already validated in prototype form, allowing MS studies at the single-molecule level that will unambiguously elucidate the details of biochemical networks that give cells their function.